To permit description of biological structures with high resolution, that is, 1-3 nm, it is customary to use a transmission electron microscope (TEM) to image the structure. The high vacuum which forms the environment for the sample in the TEM, in the order of 10.sup.-6 mbar, as well as the capability of penetrating extremely thin slivers of samples, for example 0.1 .mu.m thick, requires preparation methods for the biological test samples which do not interfere with the structure of the samples and, further, permit the samples to withstand the thin slicing as well as the vacuum.
It has been proposed to chemically fix the biological test samples for TEM use. In a first step, the samples are cross-linked in a suitable buffer solution, then dewatered or dessicated with a suitable solvent. The sample is then penetrated with monomers and polymerized. Such samples can then be sliced very thinly and examined in the TEM. It has been known for decades, however, that this chemical process affects the samples. Entire cells or portions of cells may shrink or blow or expand. Molecules are not fixed in situ, but may shift. Diffusible ions are not immobilized.
The only alternative to chemical fixation of test samples is by freezing. In this method, a first solidification step solidifies the biological sample by rapid cooling. The methods which permit investigation by a TEM are substituted by freezing with subsequent embedding, frozen etching and cutting or severing while frozen. The test samples are well immobilized by freezing and have a microstructure which is quite similar to the actual structure which the test sample has. Thus, the freezing fixation is superior to chemical fixation--see Studer et al, High Pressure Freezing comes of Age, Scanning Microscopy Supplement 3, 1989, pp. 253-269.
Biological samples have a moisture or water content of between 30-95%. Upon immobilizing by freezing, two different events may occur, in dependence on whether the freezing step is rapid or slow.
If the freezing is slow, herein considered to be several hundred degrees C. per second, ice will form and a massive phase separation will occur within the biological sample. Growing ice crystals which only consist of water molecules concentrate at their edges the materials contained within the cellular fluid, such as sugar, proteins, nucleic acids, fats, ions and the like. Test samples immobilized by freezing in this matter have segregation patterns, that is, network patterns which are very fine, within the nanometer region, or even rather coarse, that is, within the micrometer region. Such test samples do not form suitable alternatives to chemically fixed samples.
If cooling is rapid, that is at a rate of more than a million degrees C. per second, it is possible to vitrify the biological sample. At such freezing rate, there is no time for water molecules to form ice crystals. Rather, they become stiff or solidify and form a solid amorphous body. The vitrified state of water is stable below -135.degree. C. Vitrified samples maintain the desired structure which is representative of the actual structural condition, see Michel et al. (1991), J. Microsc. 163:3-18.
To obtain vitrification, it is necessary to have very high cooling rates, that is, the temperature of the sample, with respect to time, must drop rapidly. Such cooling rates can be obtained at the surface of a sample by all customary freezing methods which operate under atmospheric pressure. Within the interior of the sample, however, the cooling rate depends entirely on the physical characteristics of the object. For pure water, the maximum cooling rate in the center of a layer of 0.1 mm thickness, which is optimally cooled from both sides, is about 14,000.degree. C. per second, for a temperature drop between 0.degree. C. and -90.degree. C. This means that it is possible to vitrify only very thin samples. Results actually obtained in practice show that the test samples are frozen without interfering ice formation by only a few micrometers removed from the surface. In order to be able to vitrify thicker biological test samples, it is necessary to change the freezing characteristics of the samples. It has been proposed to add antifreeze substances, or to use high hydrostatic pressure.
Adding antifreeze substances permits vitrification with low freezing rates. However, it is necessary to first chemically cross-link biological test samples before they can be treated with antifreeze materials. The result is that the maintenance of the structure of the test sample is no better than in chemical fixation. Use of antifreeze materials, thus, is no further considered when an improved imaging of the structure, with ultra-precision is desired.
Using hydrostatic pressure which is high, in the order of 2,045 bar, lowers the melting point of water which, as well known, is 0.degree. C. at atmospheric pressure, to about -22.degree. C. Supercooling, which at atmospheric pressure is -39.degree. C., can be dropped, by pressure, to -92.degree. C., see Kanno et al. (1975), "Supercooling of water to -92.degree. C. under pressure". Science 189:880-881. It has been believed, based on theoretical considerations, that biological samples could be vitrified at a pressure of 2,045 bar at a cooling rate of several 100.degree. C. per second, see Moor H. (1987), Theory and practice of high pressure freezing. In: Steinbrecht R. A., Zierold K (eds) Cryotechniques in Biological Electron Microscopy, Berlin, Springer, 175-191.
Moor and his collaborators developed a high-pressure freezing machine in which liquid nitrogen at a temperature of -150.degree. C. is impinged on the biological sample. The samples are held in a sample carrier by two disk-like gold plates. The gold plates have a diameter of 3 mm, and a thickness of 0.6 mm, and are formed with a recess of 0.5 mm. The recess diameter is 2 mm. This prevents destruction of the probes during the rise in pressure and the cooling step.
High-pressure freezing machines are described in the referenced German Patent 1 806 741, Moor et al., and the Publication by BALZERS Union AG of Balzers, Principality of Liechenstein: "Elektronenmikroskopie--Hochdruck-Gefriermaschine HPM 010" ("Electron Microscopy--High-Pressure Freezing Machine HPM 010"). A commercially available machine provides a guarantee of a pressure rise to 2,000 bar in about 25 milliseconds. The sample is cooled immediately after reaching the 2,000 bar pressure at a cooling rate of 5,000.degree. C./sec (Moor, 1987, referred to above). The coordination of pressure rise and cooling of the sample is obtained by filling the test sample chamber with alcohol before the freezing step. Upon introduction of liquid nitrogen, the pressure rises rapidly, and the alcohol first and then the nitrogen can vent through an opening in the test chamber. Practical results have shown that there are biological samples which can be vitrified up to a thickness of about 0.150 mm. Test samples were early or young leaves of apple trees (Michel et al., 1991, supra). One can deduce therefrom that the freezing characteristics of apple tree leaves are relatively good, that is, freezing rates of several thousand .degree. C./sec., and under a pressure of 2,045 bar, are sufficient in order to obtain vitrification. Other biological samples, however, and particularly animal tissue, could not be immobilized just as well by cold. Cartilage tissue, for example, which is 80% water, is vitrified under these conditions only at the surface. The samples were 0.2 mm thick, and the vitrification depth was only about 0.02 mm. One can deduce therefrom that the physical characteristics of biological samples define the limits of possibility of vitrification. There is a need to obtain ideal cooling conditions.
The commercial high-pressure freezing machine reaches values which are not optimal for all uses and cannot be improved by simple modification. The commercial machine has a pressure rise of about 25 milliseconds and a freezing rate of 5,000.degree. C./sec. between 0.degree. C. and -50.degree. C. If the pressure period is increased by increasing the outlet from the test sample chamber, a higher throughput rate of liquid nitrogen can be obtained and a higher cooling rate will result. Such change, however, so interferes with the coordination of pressure rise and temperature drop that the biological test sample is cooled before the 2,000 bar pressure is obtained. Consequently, the sample is frozen while forming ice crystals and will not vitrify, although the cooling rate is high.